The cystic fibrosis transmembrane conductance regulator (CFTR) was the first mammalian protein implicated as a substrate for endoplasmic reticulum associated degradation (ERAD), and it has served as a model for the folding of polytopic membrane proteins. Due to its complex folding and domain assembly requirements, the majority of WT CFTR and ~100% of the common folding mutant, ?F508 CFTR, are degraded by the ubiquitin-proteasome system. We identified a novel interaction between CFTR and small heat shock proteins (sHsps), and showed that the predominant airway cell sHsp, Hsp27, selectively degrades ?F508 CFTR. This action was explained by Hsp27-mediated conjugation of CFTR with the small ubiquitin-like modifier, SUMO. Importantly, knockdown of Hsp27 or the E1 SUMO transfer enzyme increased CFTR expression 2-3 fold. Mutation of CFTR's three consensus sumoylation sites reduced WT and ?F508 CFTR expression ~80%, and also eliminated the ability of Hsp27 and the SUMO E2 to promote ?F508 CFTR degradation. These findings led to the hypothesis that Hsp27-mediated sumoylation of WT CFTR maintains domain solubility during assembly of the native structure, and that the failure to efficiently remove Hsp27 and SUMO from ?F508 CFTR targets its degradation. We will use mutagenesis, biochemical and functional assays to identify the sumoylation sites that influence the biogenesis of CFTR. The relation of Hsp27 binding and sumoylation to the conformations of WT and ?F508 NBD1 and full-length CFTR will be assessed using multiple methods: biophysical assays will include inherent tryptophan fluorescence and circular dichroism, a new enzymatic assay of NBD1 solubility, and limited proteolysis will report on compactness of NBD1 and full-length protein as a function of Hsp27 binding and SUMO modification. Progressive C-terminal CFTR truncations will explore the CFTR domain-dependence of Hsp27 binding and sumoylation, and cross-linking to Hsp27 and SUMO during CFTR translation will assess early steps in these processes. These approaches will also illuminate the relation of CFTR sumoylation to core chaperone protein interactions and to known CFTR degradation pathways. A SUMO interacting motif (SIM) is present in most sHsps, and its role in CFTR sumoylation will be evaluated. Studies of other CFTR folding mutants and of conformational mutants in other protein folding diseases will examine the generality of Hsp27- SUMO mediated stabilization/degradation. This project has provided the first evidence of sHsp involvement in CFTR biogenesis, and of SUMO conjugation to sHsp client proteins. The proposed research will clarify the molecular mechanism and the significance of this pathway in WT CFTR folding and mutant CFTR degradation, and it will provide a gateway to evaluate the significance of this system in other diseases of protein conformation. PUBLIC HEALTH RELEVANCE: This project will evaluate the hypothesis that small heat shock protein (sHsp)-mediated sumoylation initiates the degradation of the common CFTR mutant, ?F508. As a secondary hypothesis, the role of transient sHsp binding and sumoylation in the folding and assembly of wild type CFTR will be determined. This novel concept, that sHsps catalyze the transfer of SUMO to their substrates, will be evaluated as a general mechanism of sHsp chaperone function.